Transdifferentiated hepatocytes in rat pancreas.
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4-Hydroxyaminoquinoline-1-oxide↗
Biomedical subjects
D G Scarpelli
Publications and source records attributed to D G Scarpelli.
Pancreatic lesions induced by a single dose of N-nitrosobis(2-oxopropyl)amine and selection by resistance to cytotoxicity.
The experiments reported here were undertaken to ascertain whether the principle of the selection and rapid emergence of carcinogen-initiated cells based on their resistance to cytotoxicity demonstrated in rat liver is applicable to hamster pancreas. Hamsters injected with a single dose of 70 mg/kg body weight of the pancreatic carcinogen, N-nitrosobis(2-oxopropyl)amine, were subjected to pancreatic injury and regeneration induced by DL ethionine followed by methionine rescue while on a continuous daily regimen of 10 mg/kg body weight of N-nitroso-2,6-dimethylmorpholine by gavage. Randomly selected animals were killed weekly from the 4th week following the induction of regeneration through the 10th week. This carcinogenic schedule significantly decreased the time of emergence of cell injury, cell death, and proliferative, preneoplastic, and neoplastic lesions of the exocrine pancreas. Carcinoma in situ and invasive adenocarcinoma of pancreatic ducts appeared during the 7th and 8th week after induction of pancreatic regeneration, earlier than results obtained with multiple dose carcinogenesis experiments.
Animals↗
Simultaneous nuclear antigen and DNA content quantitation using paraffin-embedded colonic tissue and multiparameter flow cytometry.
The simultaneous quantitation of nuclear antigens and DNA content is presented using monoclonal antibodies and flow cytometric analysis, with paraffin-embedded human colonic pathology specimens utilized as source material. The monoclonal antibodies evaluated were shown by immunogold electron microscopy to recognize nuclear proteins preferentially associated with interchromatin (p105) and heterochromatin (p34) regions. Indirect immunofluorescence analysis of p105 revealed two distinct G1-G0 cell subpopulations in cells from normal colonic epithelium and colonic adenocarcinomas. In addition, enhanced levels of both p105 and p34 were observed in aneuploid DNA content stemlines, relative to diploid cells. Cell-sorting experiments performed on cells sorted on the basis of p105 and DNA contents reveal the capability of this method for identifying morphologically heterogeneous cell subpopulations. Other data suggest that p105 is differentially expressed in well-differentiated versus poorly differentiated tumor regions. The potential utility of this approach for the retrospective study of proliferation-associated antigens and protooncogene protein products is discussed.
Adenocarcinoma↗
Carcinogenic effect of N-nitrosobis (2-oxopropyl)amine in newborn rats.
N-nitrosobis(2-oxopropyl)amine (BOP) was administered twice weekly for a total of 11 doses to 2-day-old Fischer F-344 rats of both sexes to ascertain the spectrum of tissues sensitive to its carcinogenic effects. At 26 weeks, the following incidence of neoplasms were encountered in male and females, respectively; hepatocellular carcinoma (53 and 46%); nephroblastoma (21 and 11%); and in males gonadal stromal tumors of testis (68%). Although acidophilic and basophilic acinar cell foci were encountered in pancreas, these were few in number and microscopic. These findings indicate that in newborn Fischer rats, hepatocytes, epithelial and mesenchymal cells of the kidney, and mesenchymal cells of testis are more sensitive to BOP than those of exocrine pancreas.
Animals↗
Ductal metaplasia of human exocrine pancreas and its association with carcinoma.
Monoclonal antibodies to cell surface markers of human exocrine pancreas were used to establish the cytotypic expression of cells forming "tubular complexes" in pancreases from six adults without carcinoma and in the nontumorous pancreatic parenchyma of 16 pancreases with carcinoma. These cells manifested duct cell determinants. In general, the presence of cells with duct cell surface markers within the acini corresponded to the normal distribution of centroacinar cells in the 30 control human pancreases (from cadaveric donors); however, foci of abnormal acini were seen in these pancreases independent of or intermingled with the "tubular complexes." The acini in these abnormal areas were formed by a core of cells and cell processes that expressed duct cell determinants. They were partially surrounded by acinar cells and showed slight or no lumenal dilation. While the causative agent(s), the cell(s) of origin, and the regression and/or progression of these lesions are yet to be determined, the replacement of acini by the spectrum of lesions composed of cells with duct cell surface marker is suggested to constitute ductal metaplasia.
Animals↗
Human liver carcinogenesis: a complex problem.
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Aflatoxins↗
Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450.
The cis isomer of N-nitroso-2,6-dimethylmorpholine (NNDM), a pancreatic carcinogen for the Syrian golden hamster, is metabolized by hamster liver microsomes to yield N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) as the major product. Rabbit liver microsomes catalyze the metabolism of cis-NNDM to HPOP at a rate slower than that observed with hamster microsomes, but significantly faster than that obtained with rat microsomes. Pretreatment of rabbits with phenobarbital results in a 6-fold increase in the cis-NNDM hydroxylase activity of the rabbit microsomes to levels equal to that observed with the hamster; pretreatment of rabbits with other xenobiotics had no effect on the hydroxylation of cis-NNDM. The role of rabbit liver microsomal cytochrome P-450 in the metabolism of the cis isomer of NNDM was studied in the reconstituted system consisting of NADPH:cytochrome P-450 reductase, phospholipid, and cytochrome P-450. Cytochrome P-450LM2, which is induced by pretreatment with phenobarbital, exhibited the highest activity for the metabolism of cis-NNDM. The Vmax for the formation of HPOP was 1.78 nmol/min/nmol cytochrome P-450LM2, and the apparent Km was 360 microM. Cytochrome P-450LM3a also catalyzed the metabolism of NNDM to HPOP at a significant rate (0.25 nmol/min/nmol cytochrome P-450). Of the four other isozymes of cytochrome P-450 (forms 3b, 3c, 4, and 6) tested in the reconstituted system, only forms 3b and 3c exhibited measurable activities (approximately 0.04 nmol of HPOP formed/min/nmol cytochrome P-450). The addition of antibodies to isozyme 2 to microsomes from phenobarbital-treated rabbits resulted in approximately 95% inhibition of the metabolism of NNDM, while the addition of antibodies to LM3a inhibited NNDM metabolism by only 7%. In microsomes from untreated rabbits, inhibition by anti-LM2 and anti-LM3a antibodies was 50 and 64%, respectively. The addition of antibodies to isozyme 3a to microsomes isolated from ethanol-treated rabbits caused approximately 90% inhibition of the metabolism of NNDM. These data conclusively demonstrate that several forms of cytochrome P-450 can catalyze the metabolism of cis-NNDM and that isozymes 2 and 3a play important roles in the rabbit hepatic metabolism of NNDM to HPOP, the proximate carcinogenic metabolite.
Animals↗
Multipotent developmental capacity of cells in the adult animal.
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Animals↗
Major urinary metabolites in hamsters and rats treated with N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine.
Rats and hamsters were administered a single dose of N-[1-14C]nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), and their urinary metabolites were examined at various time intervals. In both species, urinary excretion of radiolabeled metabolites reached a plateau at 6 h following injection. At this time, 35 and 28% of the total dose was found in the urine of rats and hamsters, respectively. Separation by liquid chromatography and subsequent characterization by nuclear magnetic resonance, gas chromatography-mass spectroscopy, and infrared showed that the major metabolites in rat urine were HPOP, N-nitrosobis(2-hydroxypropyl)amine (BHP), and their glucuronic acid conjugates. The conjugates accounted for 30 and 9%, while free HPOP and BHP accounted for 42 and 16% of the total metabolites, respectively. Hamster urine, on the other hand, contained free HPOP, BHP, their glucuronic acid conjugates, and a sulfate ester of HPOP not found in rat urine. Six h following administration of HPOP, hamster urine contained BHP, BHP glucuronide, HPOP, HPOP glucuronide, and HPOP sulfate ester at levels of 35, 9, 16, 9, and 14%, respectively. These data suggest that hamsters reduce HPOP to BHP more efficiently than rats, while rats are more effective in forming their glucuronic acid conjugates. Hamsters differ significantly from rats in their capacity to form and excrete the sulfate ester of HPOP.
Animals↗
Activation of N-nitrosobis(2-oxopropyl)amine and N-nitroso(2-hydroxypropyl)-(2-oxopropyl)amine to mutagens for V79 cells by isolated hamster and rat pancreatic acinar cells.
A pancreatic acinar cell-mediated mutagenicity assay was developed as an in vitro model system to study the metabolism of N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amino (HPOP) into forms mutagenic for Chinese hamster V79 cells. Mutations at the hypoxanthine:guanine phosphoribosyltransferase locus and the Na/K ATPase locus were scored by resistance to 6-thioguanine and ouabain, respectively. The ability of both Syrian golden hamster and Fischer rat pancreatic acinar cells to convert BOP and HPOP to mutagens for V79 cells was investigated in order to examine the basis for species specificity. Acinar cells of both species were capable of activating BOP and HPOP to mutagens for V79 cells in a dose-dependent manner. In the 6-thioguanine resistance assay, rat acinar cells induced higher mutation frequencies than hamster acinar cells with both BOP and HPOP. In the ouabain resistance assay, both cell types induced equivalent levels of mutation with the respective nitrosamines. BOP was a considerably more potent mutagen than HPOP after activation by either cell type. This is consistent with the known in vivo specificity of BOP versus HPOP in the hamster pancreas and suggests that BOP may be activated to mutagenic metabolites by a pathway(s) independent from its enzymatic reduction to HPOP. The comparable abilities of rat and hamster acinar cells to convert BOP or HPOP to mutagenic forms imply that pancreatic metabolic activation alone cannot explain the difference in organotropism of BOP and HPOP in the two species.
Animals↗
Induction and origin of hepatocytes in rat pancreas.
2-[4(2,2- Dichlorocyclopropyl )phenoxy]2-methyl propionic acid (ciprofibrate), a peroxisome proliferator , induced hepatocytes in the pancreas of adult male F-344 rats when added to their diet at a dosage of 10 mg/kg body weight for 60-72 wk. These cells are morphologically indistinguishable from hepatic hepatocytes and were usually localized adjacent to islets of Langerhans with extensions into surrounding acinar tissue. A significant increase in the volume density of peroxisomes, together with immunochemically detectable amounts of two peroxisome-associated enzymes, was observed in pancreas with hepatocytes of rats maintained on ciprofibrate. Uricase-containing crystalloid nucleoids, specific for rat hepatocyte peroxisomes, were present in pancreatic hepatocytes. These structures facilitated the identification of cells with hybrid cytoplasmic features characteristic of pancreatic acinar and endocrine cells and hepatocytes. Such cells are presumed to represent a transitional state in which pancreas specific genes are being repressed while liver specific ones are simultaneously expressed. The presence of exocrine and/or endocrine secretory granules in transitional cells indicates that acinar/intermediate cells represent the precursor cell from which pancreatic hepatocytes are derived.
Animals↗
Metabolism of the cis and trans isomers of N-nitroso-2,6-dimethylmorpholine and their deuterated analogs by liver microsomes of rat and hamster.
Liver microsomes from male Syrian golden hamsters and Sprague Dawley rats metabolize the cis and trans isomers of N-nitroso-2,6-dimethylmorpholine (NNDM) to N-nitroso-(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) as the major product detectable by h.p.l.c. The rates of total metabolism are similar for both the cis and trans isomers; but the cis isomer of NNDM yields greater than 70% of the total product as HPOP while the trans isomer yields HPOP only as a minor product (20-30%) in both hamster and rat. The inability to identify other products could be attributed to alpha-hydroxylation which leads to fragmentation of NNDM and loss of tritium label to water. In order to investigate the possibility of the participation of an alpha-hydroxylation reaction, the metabolism of NNDM fully deuterated at either the 3 and 5 (alpha-d4) or the 2 and 6 (beta-d2) positions was examined and compared to the metabolism of the undeuterated compound (d0). Although the rates of metabolism of all the cis and trans derivatives of NNDM were similar (VMax = 2.13 nmol/min/mg hamster microsomal protein) as determined from measurements of substrate disappearance, the yields of HPOP were different. Maximum HPOP yields were observed with cis alpha-(d4) NNDM (93.9% of the total), followed by cis d0 NNDM (72.3%), trans alpha-(d4) NNDM (60.1%), trans d0 NNDM (30.2%), cis beta-(d2) NNDM (19.5%) and trans beta-(d2) NNDM (8.5%). These results suggest that alpha-hydroxylation is an alternative to beta-hydroxylation. Since the carcinogenic potency of the various deuterium derivatives of NNDM for the Syrian golden hamster parallels their ability to yield HPOP, beta-hydroxylation is closely related to pancreatic carcinogenesis in the hamster. Rat liver microsomal fractions showed the same patterns of HPOP formation to total metabolite yields as hamster liver microsomes with both the cis and trans isomers. However, rates of NNDM metabolism and HPOP formation were 7 times faster with hamster than with rat liver microsomes. Such a difference may be related to the failure of the cis isomer to induce pancreatic cancer in rats.
Animals↗
Studies of pancreatic carcinogenesis in different animal models.
Pancreatic carcinomas can be induced in rat, guinea pig and hamster by a variety of carcinogens. The types of neoplasms which arise vary with the species of rodent. In the rat, they consist exclusively of acinar cells, in the other species the lesions are adenocarcinomas resembling those derived from pancreatic ductules and ducts, those in hamster more so than in guinea pigs. Careful sequential studies in the guinea pig and hamster suggest that acinar cells together with ductular and duct cells are involved in the genesis of duct adenocarcinomas. In each rodent model, the acinar cell appears to be quite sensitive to continued exposure to carcinogen. In each instance, acini undergo modulation, and in the guinea pig and hamster, permanent metaplastic transformation to ductlike structures. Such cells assume an enhanced capacity for cell proliferation which persists following cessation of carcinogen treatment. Other studies suggest that adult pancreatic acinar cells possess a surprising degree of plasticity. Their involvement in the pathogenesis of neoplasms resembling pancreatic ducts is not unlike other carcinogenic sequences where extensive cell modulation and metaplasia precede and are an integral part of the neoplastic transformation.
Adenoma↗
Chemical carcinogens without mutagenic activity: peroxisome proliferators as a prototype.
Chemical carcinogens can be classified into two categories (i.e. mutagenic and non-mutagenic) on the basis of positive or negative evidence of DNA damage, mutagenicity or chromosomal aberrations in short-term test systems. Evidence indicates that carcinogenic peroxisome proliferators are negative in short-term test systems. This paper outlines approaches which may be useful in identifying a chemical carcinogen without mutagenic activity. It is conceivable that an alteration in DNA, if essential for initiation of neoplasia, may be mediated indirectly by the biological effects of nonmutagenic carcinogens.
Carcinogens↗
Metabolism of pancreatic carcinogens N-nitroso-2,6-dimethylmorpholine and N-nitrosobis(2-oxopropyl)amine by microsomes and cytosol of hamster pancreas and liver.
Liver preparations from Syrian golden hamsters catalyze the metabolism of the pancreatic carcinogen N-nitroso-2,6-dimethylmorpholine largely to N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP). This reaction is catalyzed by a mixed-function oxidase in the presence of reduced nicotinamide adenine dinucleotide phosphate and oxygen at a rate of 3.8 nmol/min/mg of protein, and it is inhibited by known cytochrome P-450-specific inhibitors. A second potent pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) is converted to HPOP by hamster liver in which two enzyme systems appear to be involved. The first is a reductase associated with microsomes which reduces BOP to HPOP in the presence of reduced nicotinamide adenine dinucleotide at a rate of 9.1 nmol/min/mg of protein. The second enzyme is a cytosolic one which catalyzes the same reaction at a slower rate (2.3 nmol/min/mg of protein) and is more effective with reduced nicotinamide adenine dinucleotide phosphate as cofactor. Based on the amount of protein in hepatic cytosol and endoplasmic reticulum, the two enzymes may be involved to a similar extent in the reduction of BOP to HPOP in the liver. Pancreas, on the other hand, lacks the microsomal reductase for BOP but contains a cytosolic enzyme which catalyzes its reduction in the presence of reduced nicotinamide adenine dinucleotide phosphate at a rate of 0.35 nmol/min/mg of protein. Since both pancreatic carcinogens N-nitroso-2,6-dimethylmorpholine and BOP are metabolized to HPOP in the liver at rates much higher than those observed in the target organ pancreas, it is suggested that the liver may play an important role in pancreatic carcinogenesis in the hamster by these compounds.
Animals↗
Augmentation of carcinogenesis by N-nitrosobis(2-oxopropyl)amine administered during S phase of the cell cycle in regenerating hamster pancreas.
Pancreatic acinar cells in various rodent species are capable of undergoing modulation to duct-like structures upon extended exposure to pancreatic carcinogens. Although the majority of malignant pancreatic neoplasms induced in rat and guinea pig are of acinar cell origin, some adenocarcinomas closely resembling those of ductal derivation have been reported. In hamster, on the other hand, carcinoma-induced duct-like modulation of acinar cells is followed exclusively by the development of ductal adenocarcinoma. The present experiments, in which the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) was administered initially to hamsters during ethionine-induced pancreatic regeneration at a time when the maximum number of acinar cells was in S phase of the cell cycle, were undertaken to ascertain the extent and nature of acinar cell response to such treatment and the pattern of tumorigenesis. BOP was also administered weekly following the cessation of regeneration for periods ranging from 1.5 to 9 weeks to achieve total doses of 120, 90, and 40 mg of BOP per kg. Controls consisted of hamsters with normal nonregenerating pancreas that were treated with BOP on identical schedules to those of the experimental groups. The largest number of carcinomas (12 in 16 animals or 75%) developed in the highest-dose test group as compared to 10 in 26 animals or 38% in its control group. The difference was statistically significant (p less than 0.05). Other groups of hamsters with regenerating and nonregenerating pancreas receiving lower doses of carcinogen had tumor incidences which were not statistically different from one another. These experiments yielded ductal adenocarcinomas exclusively, although it is of interest that the two most common benign lesions encountered in these animals were cystadenomata often lined by zymogen-containing cells and duct-like modulation of acinar cells. Finally, in some of the animals with carcinomas, nests of duct-like structures, some of which appeared to arise from acini, were lined by severely atypical epithelium with numerous mitoses. The increased incidence and exclusive development of ductal adenocarcinoma in animals to whom carcinogen was administered during pancreatic regeneration coupled with the changes noted above are interpreted as presumptive evidence that acini may be involved in the pathogenesis of pancreatic ductal adenocarcinoma in the hamster.
Animals↗
Further characterization of carcinogen-induced hepatocytelike cells in hamster pancreas.
Regenerating pancreatic cells of the Syrian hamster treated at the peak of S phase with the pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) are converted into stable cells wtih morphologic and functional characteristics that are strikingly similar to those of differentiated hepatocytes. In this article the authors further document their hepatocytelike nature. Seventy-two hours after subtotal hepatectomy, pancreatic hepatocytelike cells responded with an 8-fold increase in labeled nuclei (105.8 +/- 4.04/1000 cells) which had incorporated 3H-thymidine and a 5-fold increased mitotic index (3.8 +/- 1.5 mitoses/1000 cells), as compared with similar cells in the pancreas of control animals that had undergone sham operations. Chronic administration of phenobarbital induced a 31-fold increase in the level of aryl hydrocarbon hydroxylase (AHH) in pancreas containing such cells, as compared with normal control pancreases, and caused marked proliferation of smooth endoplasmic reticulum (SER). These cells also showed an enhanced capacity for the accumulation of iron during acute iron excess, as compared with adjacent acinar cells. Collectively, these findings support the view that carcinogen-induced cells in pancreas bear a close functional resemblance to hepatocytes.
Animals↗